To Investigate The Effect Of Micheliolide On Gouty Arthritis Based On Network Pharmacology And Molecular Docking Technology

Objective:In recent years,the prevalence of the "disease of affluence" gout continues to rise,and it has become a clinical frequentness.Gouty Arthritis is an acute attack of gout.Patients usually have red and swollen lower limb joints,severe pain,limited activity,and even joint deformity and disability in severe cases,which brings great trouble to daily life.Mulanaceae Michelia species are traditional Chinese medicinal herbs with high medicinal value.They are often used to treat rheumatic arthralgia,sore throat and abdominal distention.Recent studies have shown that Micheliolide,a sesquiterpenoid component isolated from Micheliolide,has good therapeutic effects on inflammation and cancer.Our group has also conducted a number of previous studies on the anti-inflammatory activity of Micheliolide.It has been found that Micheliolide and its derivatives can improve liver and lung injury in mice with acute peritonitis,treat inflammatory bowel disease in mice,inhibit inflammatory damage of renal tubular epithelial cells,and protect the body’s kidney function.However,whether Micheliolide has therapeutic effect on gouty arthritis has not been reported.This study aims to study the anti-gouty arthritis effect of Micheliolide,in order to provide reference for the clinical use of Micheliolide as a candidate drug for antigouty arthritis,and to expand the new application of Micheliolide in the field of inflammation.Methods:Firstly,the network pharmacology method was used to predict the core targets and signaling pathways of Micheliolide in relieving gouty arthritis,and the network diagram of "Micheliolide-targets-signaling pathways-gouty arthritis"was constructed to preliminarily clarify the mechanism of action of Micheliolide against gouty arthritis.The predicted core targets were verified by molecular docking technology,and the best target was selected.Then,a mouse model of acute gouty arthritis was constructed as the object of in vivo experimental study.The changes in the condition of acute gouty arthritis mice were observed,including gait score,inflammatory index score,swelling degree,joint pathological section,and changes in serum cytokines,to explore whether Micheliolide could have therapeutic effects on gouty arthritis mice.Finally,mouse peritoneal macrophages were used as the research objects in vitro to establish the model of LPS and MSU-induced peritoneal macrophage inflammation.The cell morphology was observed,and the samples were collected.ELISA method was used to detect the content of proinflammatory cytokines in the cell supernatant,and Western Blots were used to detect the protein expression of related signaling pathways.Results:1 Network pharmacology and molecular docking technology to predict the anti-gouty arthritis effect of Micheliolide and its mechanismNetwork pharmacology technology was used to screen 778 targets related to Micheliolide and 351 targets related to gouty arthritis through target prediction databases.A total of 58 intersection targets were obtained by drawing Venny diagram.The protein interaction network was constructed and 24 targets were identified as the core targets of Micheliolide in gouty arthritis.GO analysis identified 294 terms related to biological process,38 terms related to cell components,and 57 terms related to molecular function.KEGG analysis identified 105 signaling pathways,involving inflammation,metabolism,infection and tumor signaling pathways.Micheliolide was selected as a small molecule ligand,and the core targets screened by network pharmacology were protein receptor.Molecular docking and visual analysis were performed.The results showed that Micheliolide could bind closely to its core target through hydrogen bonds.IL-1B(IL-1β)had the highest number of hydrogen bonds and the lowest binding energy with Micheliolide.2 In vivo studies confirmed the protective effect of Micheliolide in gouty arthritis model miceA mouse model of acute gouty arthritis was constructed,and the mice were randomly divided into groups to observe the condition.The mice in the model group often lifted the injured limb when walking,and even showed a "threelegged gait".The ankle joint and foot pad were red and swollen,showing severe inflammatory reaction.Pathological sections of the ankle joint showed severe inflammatory cell infiltration and synovial swelling.The symptoms of the mice in the Micheliolide group were alleviated,and the lower limbs of the test subjects occasionally lifted off the ground and limp slightly.The joints were mildly red and swollen with bony landmarks.Pathological sections of the ankle joint showed mild to moderate inflammatory cell infiltration and reduced synovial swelling.At 6 h,12 h,24 h and 48 h after modeling,the swelling degree of ankle joint and foot pad and the content of IL-1β in serum of mice in the Micheliolide group were significantly lower than those in the model group(p<0.001,p<0.001,p<0.001),and the condition was alleviated with the increasing dose of Micheliolide.3 In vitro studies confirmed the protective effect of Micheliolide on LPS and MSU-induced peritoneal macrophage inflammation modelsMacrophages were extracted from the peritoneal cavity of the mice,and the cell inflammation models induced by LPS and MSU were established.Under the inverted microscope,the cells in the blank control group had normal morphology and smooth edges without pseudopodia.After LPS and MSU stimulation,the morphology of the cells in the inflammation model group changed,and the cells shrank and aggregated and fell off into the culture medium.The morphology of the cells in the positive control group and Micheliolide group was significantly improved compared with that in the model group.ELISA showed that compared with the inflammation model group,the level of IL-1β in the supernatant of the Micheliolide group was significantly decreased(p<0.001).Western Blots showed that compared with the inflammation model group,the expression of NLRP3 inflammasoma-related proteins,such as Caspase-1 and IL-1β,was significantly down-regulated in the Micheliolide group(p<0.001,p<0.001).Conclusions:In this study,network pharmacology and molecular docking technology were used to predict the anti-gouty arthritis effect of Micheliolide and its mechanism.The core targets of Micheliolide in gouty arthritis were screened and the main signaling pathways were predicted.Micheliolide has a protective effect on LPS and MSU-induced primary peritoneal macrophage inflammation model by regulating the NLRP3 inflammasome pathway and inhibiting the secretion of pro-inflammatory cytokine IL-1β.In vivo experiments confirmed that Micheliolide could alleviate the inflammatory response of gouty arthritis mice by inhibiting the secretion of pro-inflammatory cytokine IL-1β,which was consistent with the results predicted by network pharmacology and molecular docking technology.